This proposal aims to characterize the role of Hph1, a novel protein in Saccharomyces cerevisiae whose function is unknown, in regulating metal ion homeostasis. Hph1, and its closely related homologue, Hph2, are functionally redundant integral membrane proteins that localize to the ER. Structurally, they are similar to SNARE proteins, as they each contain a coiled-coil motif followed by a single C-terminal transmembrane domain. Yeast cells deleted for both Hph1 and Hph2 fail to growth on non-fermentable carbon sources and are sensitive to a variety of environmental conditions, including cell wall damaging agents, alkaline pH, and oxidative stress. In addition, the growth of hphl Ahph2A cells is also inhibited by a variety of cations, including Na+, Li+, Cs2+, Zn2+, Co2+, and Cd2+.,Addition of iron to the growth medium restores the growth of of hphl Ahph2A cells on non-fermentable carbon sources and elevated pH. Based on these results, we suggest a role for Hphl and Hph2 in regulating metal ion homeostasis. Our aim is to identify the mechanism by which Hph proteins regulate metal ion uptake, efflux and/or sequestration. In addition, Hphl and Hph2 interact with two components of iron transport, Fet3 and Fet5, and a known regulator of Nramp-type metal ion transporters, Bsd2, in a membrane bound yeast two-hybrid assay. We have also shown that Hph1/2 are important for the stability of two Nramp-type metal ion transporters in yeast, consistent with their interaction with Bsd2. hph1Ahph2A cells display growth defects that are not observed in mutants of Nramp-type transporters and regulators, suggesting that a broad range of proteins are affected by Hph1/2. Many proteins that facilitate ion transport, storage, and utilization in the cell are highly conserved through evolution. Altering the activity of such proteins often leads to diseases in humans. In many cases, yeast has served as a valuable model to identify and study these proteins, and particularly to study their regulation. Thus, studies of proteins like Hph1/2 that play novel, uncharacterized roles in ion homeostasis are likely to provide insights into analogous processes in human cells. As a first step to understanding Hphl function at a molecular level, we propose to (1) identify functionally important regions of Hphl and (2) to identify proteins with which it interacts using genetic, biochemical and cell biology approaches. This proposal aims to further our understanding of how metal ions are regulated. Metal ions are critical molecules needed for proper cellular function and development. Defects in metal ion transport, storage, and/or utilization are associated with diseases, such as anemia and neurological, heart, lung and liver diseases.